The need for recombinant proteins, including therapeutic proteins, is acute and there is a continuing need to improve the efficiency of production of recombinant proteins. Increasing protein yield during commercial production remains a significant challenge.
It has been reported that transient transfection with HBx plasmid can result in up-regulation of the expression of heterologous lymphotoxin alpha by Huh7 and Chang hepatoma cells. Lee et al., Biochim Biophys Acta 1741, 75-84 (2005). In addition, Chang hepatoma cells that are stably transfected with HBx can have up-regulated expression of lymphotoxin alpha.
It has also been reported that, in mammalian hepatocarcinoma suspension cells, transfection with HBx can reduce the level of E-cadherin expression in a dose-dependent manner. Lee et al., Oncogene 24, 6617-6625 (2005).
Secreted and membrane proteins typically undergo folding and other post-translational modifications in the endoplasmic reticulum (ER)-Golgi system of the host cells. The function of the unfolded protein response (UPR) signaling pathway is to sense unfolded protein levels and to adjust the protein folding and secretion capacity of the cells to environmental changes such as ER stress. Bernales et al., Annu. Rev. Cell Develop. Biology 22, 487-506 (2006). ER stress is a condition in which the capacity of the ER to fold proteins becomes saturated. IRE1 (inositol-requiring enzyme 1) and ATF6 (activating transcription factor 6) are two major sensors of unfolded proteins inside of the cells and are currently understood to be key transducers of UPR. Credle et al., Proc Natl Acad Sci USA 102, 18773-18748 (2005); Nadanaka et al., Mol. Cell. Biology 27, 1027-1043 (2007).
These metabolic pathways are activated by a number of viral genes including: a) Human cytomegalovirus 27 kDa ER-resident type I membrane glycoprotein, within the short unique region 11 (US11), which targets major histocompatibility complex (MHC) class 1 molecules for dislocation from ER to cytosol; b) nonstructural protein NS4B encoded by Hepatitis C virus (HCV), which physically interacts with CREB-RP/ATF6β and activates ATF6 and IRE1 pathways by inducing XBP1 splicing. Zheng et al., J. Microbiology 43, 529-536 (2005); and c) Hepatitis B virus 17 kDa protein within the small open reading frame X gene (HBx), which is a multifunctional transcription activator that regulates a variety of cellular events such as cell cycle, survival, and apoptosis.
IRE1, an endogenous exonuclease, can modify X-box binding protein mRNA (XBP1) to form spliced XBP1 mRNA (XBP1s), which results in a translation frame-shift, the product of which activates increased protein secretion through UPR pathway. Tigges et al., Metab. Eng. 8, 264 (2006). Tigges et al. further disclose transfection of cells with XBP1 and XBP1s to increase secretory capacity for biopharmaceutical manufacturing of secreted protein therapeutics. Tigges et al. also teach that transient transfection with XBP1s plasmid and the genes encoding secreted embryonic alkaline phosphatase (hSEAP), the secreted hormone VEGF, or secreted α-amylase can result in protein production of hSEAP, VEGF, and α-amylase, respectively.
Recombinant proteins are produced by a variety of techniques, including culturing host cells on solid substrates, on suspended microcarriers, and, for anchorage-independent cell, in non-adherent suspension culture. Adherent cultures include culture on microcarriers that can be maintained in suspension. In a suspension culture of anchorage-independent cells, cells are not attached to a substrate, but instead are maintained in suspension in a nutrient-containing medium within a suitable reaction vessel.
Various suspension methods are utilized for the production and maintenance of particular host cells. Typical suspension cell culturing methods for microcarriers and for anchorage-independent cells use shakers, rollers, stirrers, or air-lift systems that agitate a cell culturing medium and cells in culture or in bioreactors. Aeration, temperature, rate of agitation and like conditions can be controlled and adjusted to provide conditions favorable to growth of the culture and high production of recombinant protein.